ES2628334T3 - CwpV cell wall protein (CD0514) as a diagnostic marker for Clostridium difficile 027 ribotype - Google Patents

Abstract

A method for diagnosing a patient with an infection by strains of C. difficile of ribotype 027, which method comprises: obtaining a fecal sample from the patient; determine that the patient has a C. difficile infection; and determining whether the patient is infected with ribotype 027 C. difficile strains by determining the presence of a ribotype 027-specific CwpV C-terminal domain antigen in the fecal sample.

Description

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DESCRIPTION

CwpV cell wall protein (CD0514) as a diagnostic marker for Clostridium difficile 027 ribotype Background of the invention

Clostridium difficile (C. difficile) is the most common common cause of nosocomial diarrhea and accounts for approximately 3 million cases of diarrhea annually in the United States. Risk factors for C. difficile infection (CDI) include exposure to antibiotics, old age, and residence in hospitals or long-term care facilities. The symptoms of CDI range from mild diarrhea to pseudomembranous colitis and toxic megacolon. The average cost of treatment is approximately $ 10,000 per case. The death rate of CDI increased from 5.7 deaths per million population in 1999 to 23.7 deaths per million population in 2004 due to the appearance of outbreaks of hypervirulent strains. The 027 ribotype strain contributed significantly to the higher incidence of CDI. The precise differentiation of the outbreak of the 027 ribotype strain from other C. difficile strains can facilitate decision making for treatment options.

Brief description of the drawings

Illustrative embodiments of the invention are described in detail below with reference to the attached figures of the drawings, wherein:

Fig. 1 depicts a comparison between anti-CwpV ELISA and PCR ribotyping methods, in accordance with the embodiments of the invention;

Fig. 2 represents that antibodies generated against the specific region of 027 of CwpV only recognize ribotype 027 of C. difficile, in accordance with the embodiments of the invention;

Fig. 3 represents the interpretation of the QUIK CHEK® results of the Anti-CwpV; Y

Fig. 4 depicts a comparison between QUIK CHEK® of the anti-CwpV and the ribotyping methods by PCR, in accordance with the embodiments of the invention.

Detailed description of the invention

Embodiments of the present invention are directed to assay methods for differentiating strains of C. difficile in patients based on the presence of a new specific antigen marker of a ribotype, Cell Wall Protect V (CwpV). The specific CwpV of a ribotype can be used in an immunoassay for the detection of C. difficile ribotypes, especially the outbreak of strain 027. In embodiments, specific CwpV antibodies of the anti-strain are used in immunoassays for highly sensitive identification. of strain 027 of C. difficile.

Several methods of typing have been developed to study the genetic relationship between strains of C. difficile and the association between strains of C. difficile and the severity of the disease. These methods include serotyping, toxinotyping, pulsed field gel electrophoresis (PFGE) and PCR ribotyping. Ribotyping by PCR is relatively easy to perform, reproducible, and is one of the most discriminatory methods to differentiate strains of C. difficile.

US Patent 2011/020845 is an example of a method for specifically detecting strain 027 based on a specific antigen of strain 027 on the TcdB toxin.

Since the late 1990s, a 5-fold increase in CDI has been observed and the spread of a hypervirulent strain is suspected to contribute to the outbreak. This strain of the strain was characterized as toxinotype III, pulsed field gel electrophoresis of North America type 1 (NAP1), endonuclease restriction analysis group I (BI) and ribotype 027 PCR. Several ribotype 027 characteristics contribute to improve virulence of strains of ribotype 027. In addition, of toxins A and B, strains 027 express an additional binary toxin that can contribute to the severity of the disease. Strains 027 also contain a deletion of 18 base pairs and a mutation of the reading frame in the tcdC gene, which is the repressor of toxin production. Reading frame mutation can cause an increase in toxin production. Historical 027 strains isolated in France in 1988 were susceptible to fluoroquinolones and erythromycin but mutations in gyrA / B and 23s RNA genes made ribbons 027 in recent outbreaks with resistance to both antibiotics. Altered surface proteins with higher affinity to human intestinal epithelial cells may also contribute to increased virulence.

Infections with 027 strains were associated with more severe results in some studies. The dramatic alteration of the intestinal microbiota by strain 027 is likely to contribute to its high pathogenicity. However, other studies indicated the absence of correlation between the ribotype of the infecting strain and the outcome of the patients. The controversies could have resulted from the population studied and the methods used in the statistical analysis.

Traditional antibiotics of choice for the treatment of IDU are metronidazole and vancomycin. For patients with mild to moderate CDI, metronidazole and vancomycin are equally effective. For patients with

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Severe CDI caused by non-027 strains, vancomycin produced better results. A high rate of relapses was observed in patients treated with metronidazole or vancomycin. In May 2010, a new drug called Fidaxomycin (Dificid; Optimer Pharmaceuticals) was approved by the U. S. Food and Drug Administration (FDA) for the treatment of diarrhea associated with C. difficile in adults. Fidaxomycin is a narrow-spectrum antibiotic against gram-positive anaerobes. Fidaxomycin resistance was found in Bacteroides spp., Aerobic and facultative gram-negative bacilli and anaerobic gram-negative bacilli. Fidaxomycin is equally effective against 027 and non-027 strains of C. difficile. The use of broad-spectrum antibiotics disturbs the intestinal flora, which may contribute to the recovery of C. difficile infection. Recent studies indicate that treatment with Fidaxomycin resulted in a lower rate of recapture in patients infected with non-027 strains compared to treatment with Vancomycin. Therefore the differentiation between strains 027 and non-027 can facilitate the decision making of doctors in terms of treatment options.

The PCR ribotyping method requires the isolation of colonies of C. difficile, the isolation of DNA from the culture originated from pure colonies, the amplification of DNA by PCR, and gel electrophoresis. The complicated and time-consuming procedure is not practical in the chemical laboratories. Commercially available molecular tests use sequence variation in the tcdC gene. PCR-based tests are expensive and not specific because mutations in the tcdC gene are also present in other ribotypes. Antibody-based tests, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow assays, are rapid and cost-effective tests for the detection of pathogen-specific antigens. In embodiments, the specific antigen (s) of strain 027 are used as a diagnostic marker (s) for the detection of 027 strains in immunoassays.

C. difficile cells have a surface layer (layer S) outside the peptidoglycan layer. The S layer is present both in vegetative cells and in C. difficile spores. Proteins within the S layer mediate host-pathogen interactions. Several surface proteins of C. difficile bind to gastrointestinal tissues and are potential colonization factors. The proteins associated with the S layer contain two domains: a conserved cell wall binding domain and a variable domain that specifies the function of that particular protein. Twenty-eight cell wall proteins (CWPs) were predicted in the genome of the 630 sequenced strain. The variable domains of CWPs deltas are considered potential antigen markers for the identification of the C. difficile strain.

CwpV (CD0514) is a member of the CWPs and consists of an N-terminal region with an assumed cell wall binding domain and a C-terminal domain that can promote aggregation. Reynolds et al., PLOS Pathogens, vol. 7, no. 4, 2011 describe the C-terminal part of CwpV in strain 027.

CwpV is secreted in the culture medium by several 027 strains. The C-terminal domain of CwpV is highly variable among C. difficile ribotypes. The specific amino acid sequences of the C-terminal repeats found in the CwpV protein in ribotype 027 are not represented in the genome of any other strain sequenced to date. In embodiments, the specific region of the CwpV ribotype can be used as a diagnostic marker for 027 strains.

The following are examples of procedures that have been used to establish the preferred assays in accordance with the embodiments of the present invention. The following examples are merely illustrative and are not presented by way of limitation.

Example 1

A specific C-terminal peptide of the 027 CwpV ribotype was expressed in E. Coli using recombinant DNA techniques. Polyclonal antibodies against recombinant CwpV were generated in goats. An ELISA was developed using anti-CwpV polyclonal antibodies as capture antibodies and anti-CwpV polyclonal antibodies conjugated to horseradish peroxidase (HRP) as detection antibodies.

Seventy-two (72) chlorine human fecal samples that were positive for C. difficile were tested using this anti-CwpV ELISA. The sensitivity and specificity of the anti-CwpV ELISA for the detection of strains 027 of C. difficile were calculated using the PCR ribotyping method as a gold standard. The cut-off point of this ELISA was set to be an absorbance of 0.080 measured at a double wavelength (OD450 / 620nm).

Results

With reference to Fig. 1, the results of the anti-CwpV ELISA in 72 human fecal samples were compared with the results of the PCR ribotyping method. The anti-CwpV ELISA detected the presence of strain 027 of C. difficile in 30 of the 35 positive samples of 027 determined by the PCR ribotyping method. The sensitivity and specificity of anti-CwpV ELISA are each 86%.

Example 2

A specific C-terminal peptide of CwpV 027 ribotype was expressed in E. Coli. Polyclonal antibodies against recombinant CwpV were generated in goats. An ELISA was developed using polyclonal anti antibodies

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CwpV as capture antibodies and polyclonal anti CwpV antibodies conjugated to horseradish peroxidase (HRP) as detection antibodies.

Thirty-four (34) strains of C. difficile were inoculated in a broth of cerebral heart infusion (BHI, Oxoid). The cultures were grown at 37 ° C overnight under anaerobic conditions.

The C. difficile strains in BHI cultures were diluted 1:20 in phosphate buffered saline (PBS) and tested with ELISA as described above.

Results

As shown in Fig. 2, C. difficile cultures representing thirty-four (34) different ribotypes were tested with anti CwpV ELISA using anti-027 specific antibodies. The 027 ribotype strain was the only ribotype that was detected in this ELISA. The thirty-three (33) cultures of C. difficile no 027 were not detected by this ELISA. The cut-off point of this ELISA was set to be an absorbance of 0.080 measured at a double wavelength (OD450 / 620nm).

Example 3

A specific C-terminal peptide of CwpV 027 ribotype was expressed in E. Coli. Polyclonal antibodies against recombinant CwpV were generated in goats. A QUIK CHEK® device was developed using immobilized anti-CwpV polyclonal antibodies such as the microfiber glass membrane test line as capture antibodies and anti-CwpV polyclonal antibodies conjugated to horseradish peroxidase (HRP) as detection antibodies. Polyclonal rabbit anti HRP antibodies were immobilized on the microfiber glass membrane to form a positive control line.

One hundred ninety-five (195) samples were tested using this QUIK CHEK® anti CwpV. Chloric stool samples were diluted in a sample diluent, mixed with anti-CwpV polyclonal antibodies conjugated with HRP and applied to the microfiber glass membrane with capture antibodies immobilized through the sample well. The conjugate mixture of the sample flowed through the microfiber glass membrane by capillary action. The device was incubated at room temperature for fifteen (15) minutes to allow the capture of the CwpV antigen in the samples by the anti-CwpV antibodies immobilized in the test line. The excess polyclonal anti-CwpV antibodies conjugated with HRP were captured by rabbit anti-HRP polyclonal antibodies immobilized in the control line. After rapid washing of the membrane and the addition of HRP substrate through the reaction window, the device was incubated at room temperature for ten minutes before recording the results.

With reference to Fig. 3, a positive result in QUIK CHEK® anti CwpV is indicated by two blue lines: the control line ("C") and the test line ("T"). A negative result in QUIK CHEK® anti CwpV is indicated by a single blue line on the control side ("C") of the reaction window without a visible test line on the "T" side of the reaction window. The result is interpreted as invalid when the control line ("C") is not visible.

Results

One hundred ninety-five (195) faecal samples were tested in the QUIK CHEK® anti CwpV, in the tissue culture cytotoxicity test, and in C. difficile ribotypes isolated from samples containing C. difficile toxigenico were determined by ribotyping method by PCR. As shown in Figure 4, among the 195 fecal samples tested, one hundred seventy-three (173) samples were determined not to contain C. difficile toxigenico by the tissue culture cytotoxicity test. All samples that were negative by the tissue culture method were negative in QUIK CHEK® anti CwpV. Among the twenty-two (22) samples determined to contain C. difficile by the tissue culture cytotoxicity method, twelve (12) were identified as ribotype 027 by PCR ribotyping. Ten (10) of the twelve samples showed a positive reaction when QUIK CHEK® anti CwpV was used. None of the C. difficile no. 027 samples were detected by QUIK CHEK® anti CwpV. Using the PCR ribotyping method as a gold standard, the sensitivity and specificity of QUIK CHEK® anti CwpV were calculated to be 83% and 100% respectively.

In summary, embodiments of the present invention provide CwpV as a diagnostic marker for detecting ribotype 027 of C. difficile in stool samples and in cultures. The present invention has been described in relation to particular embodiments that are intended in all aspects to be illustrative rather than restrictive. Alternative embodiments will be apparent to those skilled in the art to which the present invention belongs without departing from its scope.

From the above, it will be seen that this invention is well adapted to achieve all the purposes and objectives set forth above along with other advantages that are obvious and that are inherent to the method. It will be understood that certain features and sub-combinations of the invention are useful and can be used without reference to other features and sub-combinations. This is contemplated and is within the scope of the claims.

Claims (9)

10 fifteen twenty 1. A method for diagnosing a patient with an infection by C. difficile strains of ribotype 027, a method comprising: obtaining a stool sample from the patient; determine that the patient has a C. difficile infection; and determine if the patient has an infection by strains of C. difficile of ribotype 027 by determining the presence of an antigen of the C-terminal domain of specific CwpV of ribotype 027 in the stool sample. 2. The method of claim 1, wherein determining the presence of the antigen of the C-terminal domain of CwpV specific for ribotype 027 comprises determining the presence of the antigen of the C-terminal domain of CwpV specific for ribotype 027 using an immunoassay. 3. The method of claim 2, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or an immunochemical reaction in a membrane. 4. The method of claim 2, wherein the immunoassay uses one or more monoclonal, polyclonal or recombinant anti-ribotype 027 CwpV antibodies as capture antibodies. 5. The method of claim 2, wherein the immunoassay uses polyclonal or monoclonal anti-ribotype 027 CwpV antibodies conjugated to HRP. 6. The method of claim 2, wherein the immunoassay comprises one or more antibodies against CwpV directed against native CwpV produced by C. difficile or CwpV derivatives by recombinant DNA expression in E. Coli. 7. The method of claim 1, wherein determining the presence of C. difficile of ribotype 027 further comprises identifying the presence of a DNA fragment encoding the specific CwpV of ribotype 027 using nucleic acid amplification assays. Anti-CwpV ELISA PCR Ribotyping

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FIG. one.

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 Anti CwpV ELISA reaction

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FIG. 2. Reaction window Sample well image 1 control test FIG. image2 Not valid

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FIG. 4